The overall objective of this project is to characterize two fundamental aspects of the biology of endorphins: (I) the enzymatic activity involved in the synthesis of enkephalins, and (II) the biochemical mechanisms of opiate tolerance and dependence. I. We have found peptidases contained in the bovine chromaffin granules that convert the putative adrenal medulla enkephalin precursor into methionine-and leucine-enkephalin. In addition to further the purification and characterization of these enzymes, we will search for inhibitors and raise antibodies to study the specificity and distribution of the enzymes in brain and other organs. II. We will study the acute and chronic changes produced by opiates and opiate peptides on the levels of guanine and adenine nucleotides in cultured cell lines which are known to be model systems for studying the biochemical signals which may mediate the effects of opiates. Using methods we have developed for the simultaneous measurement of all the guanine and adenine nucleotides, we will determine their relative concentrations after acute and chronic opiate treatment and naloxone challenge. Patterns of change will identify the enzyme responsible for the adaptation to chronic opiate exposure. After identification, the enzyme will be measured under a variety of conditions. Studies will be validated by searching for similar changes in the striatum of rats after acute and chronic opiate treatment.